The pulmonary microvascular endothelium forms a barrier limiting the movement of blood components into the underlying tissue. Barrier disruption leads to increased permeability that contributes to multiple pathologies including sepsis and acute respiratory distress syndrome. Cell-cell adhesions critically maintain this barrier and in turn are governed by intracellular cAMP concentrations. However, cAMP signaling specificity is encoded in the spatial and temporal fluctuations of this second messenger. Endogenous transmembrane adenylyl cyclase (AC) 6 synthesizes an endothelial barrier protective near-membrane cAMP-pool which is targeted to physiologically relevant effectors by phosphodiesterase (PDE) 4 confined to the membrane by spectrin. In contrast, the pathogenic bacteria Pseudomonas aeruginosa inserts a soluble AC, ExoY, into pulmonary microvascular endothelial cells (PMVECs) and generates a cytosolic cAMP pool that disrupts the barrier. Thus, this proposal tests the overall Hypothesis that endogenous transmembrane ACs generate a membrane cAMP pool does not saturate the cytosolic compartment while soluble ACs generate a cytosolic cAMP pool that does not reach the plasma membrane. Specific aims test the related hypotheses that (1) endogenous transmembrane ACs generate a membrane-to-cytosol cAMP gradient while soluble ACs disrupt this gradient and (2) plasma membrane localized PDE4(D4) activity prevents the spread of cAMP away from the plasma membrane domain. cAMP fluctuations in live PMVECs in response to endogenous- or ExoY-AC activity will be determined using electrophysiological techniques to detect near-membrane cAMP concentrations while the extent of second messenger diffusion will be demonstrated through FRET studies. Completion of these studies will give a clearer understanding of how endogenous ACs are barrier protective while bacterial toxins, such as ExoY of P. aeruginosa, disrupt the endothelial barrier. [unreadable] [unreadable] [unreadable] [unreadable]